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  • 99
    New England Biolabs mlui
    Shuttle mutagenesis strategy used to construct vLIP mutant MDVs and revertants in the pRB-1B BAC. A 4.268-kb fragment of the Md5 strain of MDV, containing the vLIP gene and portions of neighboring genes, was cloned into pST76K-SR, a RecA-based shuttle vector. In step 1 (labeled arrow), an in-frame deletion of vLIP amino acids 256 to 426 was incorporated into the pRB-1B BAC by shuttle mutagenesis using a pST76K-SR-based shuttle vector bearing the same deletion <t>(ΔMluI-SpeI),</t> yielding Δ vLIP . In parallel (step 2), an alanine point mutant of the vLIP serine nucleophile position ( vLIP S307A) was incorporated into pRB-1B. As depicted in steps 3 and 4, shuttle mutagenesis was performed on the Δ vLIP BAC to generate C-terminally FLAG tagged vLIP ( vLIP *-rev) and native vLIP ( vLIP -rev) revertants. Relevant features of the DNA fragment used and modified in shuttle mutagenesis procedures are labeled accordingly. A double-headed arrow represents lipase homology in the vLIP ORF, a white X represents the location of the S307A change, a FLAG epitope tag is represented as a labeled asterisk, and the <t>MluI</t> and SpeI sites which were used to remove the lipase homology region of vLIP are labeled accordingly.
    Mlui, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1394 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher mlui
    Shuttle mutagenesis strategy used to construct vLIP mutant MDVs and revertants in the pRB-1B BAC. A 4.268-kb fragment of the Md5 strain of MDV, containing the vLIP gene and portions of neighboring genes, was cloned into pST76K-SR, a RecA-based shuttle vector. In step 1 (labeled arrow), an in-frame deletion of vLIP amino acids 256 to 426 was incorporated into the pRB-1B BAC by shuttle mutagenesis using a pST76K-SR-based shuttle vector bearing the same deletion <t>(ΔMluI-SpeI),</t> yielding Δ vLIP . In parallel (step 2), an alanine point mutant of the vLIP serine nucleophile position ( vLIP S307A) was incorporated into pRB-1B. As depicted in steps 3 and 4, shuttle mutagenesis was performed on the Δ vLIP BAC to generate C-terminally FLAG tagged vLIP ( vLIP *-rev) and native vLIP ( vLIP -rev) revertants. Relevant features of the DNA fragment used and modified in shuttle mutagenesis procedures are labeled accordingly. A double-headed arrow represents lipase homology in the vLIP ORF, a white X represents the location of the S307A change, a FLAG epitope tag is represented as a labeled asterisk, and the <t>MluI</t> and SpeI sites which were used to remove the lipase homology region of vLIP are labeled accordingly.
    Mlui, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1265 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega mlui
    Electrophoresis of six constructs digested with <t>Mlu</t> I and Xho I. M1: 100 bp DNA ladder marker, M2: 1 kb DNA ladder marker, Lane1: pCOLH 1 0.1, lane2: pCOLH 1 0.27, lane3: pCOLH 1 0.5, Lane4: pCOLH 1 0.9, Lane5: pCOLH 1 1.5, Lane6: pCOLH 1 2.5. Six recombinant plasmids
    Mlui, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 1270 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    mlui  (TaKaRa)
    99
    TaKaRa mlui
    Specificity of telomere interactions analyzed by the 3C procedure. ( A ) Physical map of pLUS892L and the predicted cross-linked, ligated products. On pLUS892L (top), the positions of the L and R primers and their distances to the nearest <t>MluI</t> (‘Ml’) sites are indicated (in kb). On the expected intramolecular (middle) and intermolecular (bottom) products, the expected PCR products and their sizes (in kb) are indicated. Cross-linking is depicted by the dashed arcs. The other symbols are as in Figure 1 . ( B ) Detection of intermolecular and intramolecular cross-linking. S. lividans 3200/pLUS892L mycelium was treated with DSS, and lysed by protoplasting and osmotic shock. <t>DNA</t> in the lysate was digested with MluI, and ligated at two different concentrations (top panel, 20-µl ligation reaction; bottom panel, 1-ml ligation reaction) followed by LM-PCR using the R, L or both primers (‘R + L’) in the absence (‘0%’) or presence (‘10%’) of DMSO. The PCR products were analyzed by gel electrophoresis. The sizes of the PCR products are indicated (in kb). ( C ) Detection of intramolecular cross-linking after GnHCl centrifugation. Cross-linking and detection were performed as in B except for an additional GnHCl–CsCl gradient centrifugation step (right half) DNA before cross-linking. The volume of the ligation reaction was 1 ml.
    Mlui, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 419 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs mlui hf
    Specificity of telomere interactions analyzed by the 3C procedure. ( A ) Physical map of pLUS892L and the predicted cross-linked, ligated products. On pLUS892L (top), the positions of the L and R primers and their distances to the nearest <t>MluI</t> (‘Ml’) sites are indicated (in kb). On the expected intramolecular (middle) and intermolecular (bottom) products, the expected PCR products and their sizes (in kb) are indicated. Cross-linking is depicted by the dashed arcs. The other symbols are as in Figure 1 . ( B ) Detection of intermolecular and intramolecular cross-linking. S. lividans 3200/pLUS892L mycelium was treated with DSS, and lysed by protoplasting and osmotic shock. <t>DNA</t> in the lysate was digested with MluI, and ligated at two different concentrations (top panel, 20-µl ligation reaction; bottom panel, 1-ml ligation reaction) followed by LM-PCR using the R, L or both primers (‘R + L’) in the absence (‘0%’) or presence (‘10%’) of DMSO. The PCR products were analyzed by gel electrophoresis. The sizes of the PCR products are indicated (in kb). ( C ) Detection of intramolecular cross-linking after GnHCl centrifugation. Cross-linking and detection were performed as in B except for an additional GnHCl–CsCl gradient centrifugation step (right half) DNA before cross-linking. The volume of the ligation reaction was 1 ml.
    Mlui Hf, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 127 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher fastdigest mlui
    Specificity of telomere interactions analyzed by the 3C procedure. ( A ) Physical map of pLUS892L and the predicted cross-linked, ligated products. On pLUS892L (top), the positions of the L and R primers and their distances to the nearest <t>MluI</t> (‘Ml’) sites are indicated (in kb). On the expected intramolecular (middle) and intermolecular (bottom) products, the expected PCR products and their sizes (in kb) are indicated. Cross-linking is depicted by the dashed arcs. The other symbols are as in Figure 1 . ( B ) Detection of intermolecular and intramolecular cross-linking. S. lividans 3200/pLUS892L mycelium was treated with DSS, and lysed by protoplasting and osmotic shock. <t>DNA</t> in the lysate was digested with MluI, and ligated at two different concentrations (top panel, 20-µl ligation reaction; bottom panel, 1-ml ligation reaction) followed by LM-PCR using the R, L or both primers (‘R + L’) in the absence (‘0%’) or presence (‘10%’) of DMSO. The PCR products were analyzed by gel electrophoresis. The sizes of the PCR products are indicated (in kb). ( C ) Detection of intramolecular cross-linking after GnHCl centrifugation. Cross-linking and detection were performed as in B except for an additional GnHCl–CsCl gradient centrifugation step (right half) DNA before cross-linking. The volume of the ligation reaction was 1 ml.
    Fastdigest Mlui, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore mlu
    Specificity of telomere interactions analyzed by the 3C procedure. ( A ) Physical map of pLUS892L and the predicted cross-linked, ligated products. On pLUS892L (top), the positions of the L and R primers and their distances to the nearest <t>MluI</t> (‘Ml’) sites are indicated (in kb). On the expected intramolecular (middle) and intermolecular (bottom) products, the expected PCR products and their sizes (in kb) are indicated. Cross-linking is depicted by the dashed arcs. The other symbols are as in Figure 1 . ( B ) Detection of intermolecular and intramolecular cross-linking. S. lividans 3200/pLUS892L mycelium was treated with DSS, and lysed by protoplasting and osmotic shock. <t>DNA</t> in the lysate was digested with MluI, and ligated at two different concentrations (top panel, 20-µl ligation reaction; bottom panel, 1-ml ligation reaction) followed by LM-PCR using the R, L or both primers (‘R + L’) in the absence (‘0%’) or presence (‘10%’) of DMSO. The PCR products were analyzed by gel electrophoresis. The sizes of the PCR products are indicated (in kb). ( C ) Detection of intramolecular cross-linking after GnHCl centrifugation. Cross-linking and detection were performed as in B except for an additional GnHCl–CsCl gradient centrifugation step (right half) DNA before cross-linking. The volume of the ligation reaction was 1 ml.
    Mlu, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 82 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    OriGene mlui sites
    Specificity of telomere interactions analyzed by the 3C procedure. ( A ) Physical map of pLUS892L and the predicted cross-linked, ligated products. On pLUS892L (top), the positions of the L and R primers and their distances to the nearest <t>MluI</t> (‘Ml’) sites are indicated (in kb). On the expected intramolecular (middle) and intermolecular (bottom) products, the expected PCR products and their sizes (in kb) are indicated. Cross-linking is depicted by the dashed arcs. The other symbols are as in Figure 1 . ( B ) Detection of intermolecular and intramolecular cross-linking. S. lividans 3200/pLUS892L mycelium was treated with DSS, and lysed by protoplasting and osmotic shock. <t>DNA</t> in the lysate was digested with MluI, and ligated at two different concentrations (top panel, 20-µl ligation reaction; bottom panel, 1-ml ligation reaction) followed by LM-PCR using the R, L or both primers (‘R + L’) in the absence (‘0%’) or presence (‘10%’) of DMSO. The PCR products were analyzed by gel electrophoresis. The sizes of the PCR products are indicated (in kb). ( C ) Detection of intramolecular cross-linking after GnHCl centrifugation. Cross-linking and detection were performed as in B except for an additional GnHCl–CsCl gradient centrifugation step (right half) DNA before cross-linking. The volume of the ligation reaction was 1 ml.
    Mlui Sites, supplied by OriGene, used in various techniques. Bioz Stars score: 89/100, based on 75 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher mlui hindiii site
    Specificity of telomere interactions analyzed by the 3C procedure. ( A ) Physical map of pLUS892L and the predicted cross-linked, ligated products. On pLUS892L (top), the positions of the L and R primers and their distances to the nearest <t>MluI</t> (‘Ml’) sites are indicated (in kb). On the expected intramolecular (middle) and intermolecular (bottom) products, the expected PCR products and their sizes (in kb) are indicated. Cross-linking is depicted by the dashed arcs. The other symbols are as in Figure 1 . ( B ) Detection of intermolecular and intramolecular cross-linking. S. lividans 3200/pLUS892L mycelium was treated with DSS, and lysed by protoplasting and osmotic shock. <t>DNA</t> in the lysate was digested with MluI, and ligated at two different concentrations (top panel, 20-µl ligation reaction; bottom panel, 1-ml ligation reaction) followed by LM-PCR using the R, L or both primers (‘R + L’) in the absence (‘0%’) or presence (‘10%’) of DMSO. The PCR products were analyzed by gel electrophoresis. The sizes of the PCR products are indicated (in kb). ( C ) Detection of intramolecular cross-linking after GnHCl centrifugation. Cross-linking and detection were performed as in B except for an additional GnHCl–CsCl gradient centrifugation step (right half) DNA before cross-linking. The volume of the ligation reaction was 1 ml.
    Mlui Hindiii Site, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Addgene inc mlui digested paav ef1α dio mcherry
    CRISPR-Cas9-mediated crh gene disruption in CRH PVN neurons of CRH::Cre-Cas9 mice. (A) Generation of CRH::Cre-Cas9 mice. (B) Schematic of AAV sgCRH12 vector design. (C) Schematic of AAV-DJ-U6- <t>sgControl/sgCRH12-EF1α-DIO-mCherry</t> to unilateral PVN of CRH::Cre-Cas9 mice (n = 3, AP: −0.90 mm, ML: ±0.30 mm, DV: −4.5 + -4.8 mm, 0.3 μl each depth). (D) Representative slices from CRH::Cre-Cas9 mice injected with AAV vectors carrying sgControl or sgCRH12. Note the intact CRH staining on the contralateral side without viral infection in the sgCRH12 panel.
    Mlui Digested Paav Ef1α Dio Mcherry, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Shuttle mutagenesis strategy used to construct vLIP mutant MDVs and revertants in the pRB-1B BAC. A 4.268-kb fragment of the Md5 strain of MDV, containing the vLIP gene and portions of neighboring genes, was cloned into pST76K-SR, a RecA-based shuttle vector. In step 1 (labeled arrow), an in-frame deletion of vLIP amino acids 256 to 426 was incorporated into the pRB-1B BAC by shuttle mutagenesis using a pST76K-SR-based shuttle vector bearing the same deletion (ΔMluI-SpeI), yielding Δ vLIP . In parallel (step 2), an alanine point mutant of the vLIP serine nucleophile position ( vLIP S307A) was incorporated into pRB-1B. As depicted in steps 3 and 4, shuttle mutagenesis was performed on the Δ vLIP BAC to generate C-terminally FLAG tagged vLIP ( vLIP *-rev) and native vLIP ( vLIP -rev) revertants. Relevant features of the DNA fragment used and modified in shuttle mutagenesis procedures are labeled accordingly. A double-headed arrow represents lipase homology in the vLIP ORF, a white X represents the location of the S307A change, a FLAG epitope tag is represented as a labeled asterisk, and the MluI and SpeI sites which were used to remove the lipase homology region of vLIP are labeled accordingly.

    Journal: Journal of Virology

    Article Title: vLIP, a Viral Lipase Homologue, Is a Virulence Factor of Marek's Disease Virus

    doi: 10.1128/JVI.79.11.6984-6996.2005

    Figure Lengend Snippet: Shuttle mutagenesis strategy used to construct vLIP mutant MDVs and revertants in the pRB-1B BAC. A 4.268-kb fragment of the Md5 strain of MDV, containing the vLIP gene and portions of neighboring genes, was cloned into pST76K-SR, a RecA-based shuttle vector. In step 1 (labeled arrow), an in-frame deletion of vLIP amino acids 256 to 426 was incorporated into the pRB-1B BAC by shuttle mutagenesis using a pST76K-SR-based shuttle vector bearing the same deletion (ΔMluI-SpeI), yielding Δ vLIP . In parallel (step 2), an alanine point mutant of the vLIP serine nucleophile position ( vLIP S307A) was incorporated into pRB-1B. As depicted in steps 3 and 4, shuttle mutagenesis was performed on the Δ vLIP BAC to generate C-terminally FLAG tagged vLIP ( vLIP *-rev) and native vLIP ( vLIP -rev) revertants. Relevant features of the DNA fragment used and modified in shuttle mutagenesis procedures are labeled accordingly. A double-headed arrow represents lipase homology in the vLIP ORF, a white X represents the location of the S307A change, a FLAG epitope tag is represented as a labeled asterisk, and the MluI and SpeI sites which were used to remove the lipase homology region of vLIP are labeled accordingly.

    Article Snippet: In order to construct a shuttle vector to incorporate an in-frame deletion of amino acids 256 to 428 of the vLIP open reading frame, a 519-bp fragment was released from the pEco4.268 plasmid using MluI and SpeI, and overhangs were filled in using T4 DNA polymerase (NEB).

    Techniques: Mutagenesis, Construct, BAC Assay, Clone Assay, Plasmid Preparation, Labeling, Modification, FLAG-tag

    (a) Location of vLIP relative to other genes in the MDV genome. The vLIP open reading frame is drawn to scale; a bar representing 1 kb is drawn and labeled. The terminal repeat long (TR L ) region, the U L region, and an a-like sequence (double-headed arrow) are also indicated. Several MDV-1-specific ORFs neighboring the vLIP gene are displayed, as are the conserved genes U L 1 to U L 3. The gene organization of the vLIP transcript is shown in more detail below, also drawn to scale. A 500-bp bar is shown, and the TATA box, putatively used to initiate vLIP transcripts, as well as the poly(A) site and poly(A) signal, is labeled. MluI and SpeI restriction sites flanking the lipase homology region, indicated by a double-headed arrow, are also labeled. (b) The vLIP transcript as visualized by Northern blotting. In the left panel, a Northern blot was probed with a single-stranded riboprobe antisense to vLIP mRNA. RNA samples were derived from MSB-1 tumor cells which had been either treated for 24 h in the presence of sodium butyrate to induce virus replication or left untreated, as indicated. PFA was also used where indicated, to show the sensitivity of vLIP transcription to inhibition of DNA replication. To the right, RNA samples used in Northern blotting were separated on denatured agarose gels, stained with ethidium bromide, and imaged to verify that RNA integrity and quantity were comparable across all samples tested. An RNA marker (Ambion) was included to determine molecular weights.

    Journal: Journal of Virology

    Article Title: vLIP, a Viral Lipase Homologue, Is a Virulence Factor of Marek's Disease Virus

    doi: 10.1128/JVI.79.11.6984-6996.2005

    Figure Lengend Snippet: (a) Location of vLIP relative to other genes in the MDV genome. The vLIP open reading frame is drawn to scale; a bar representing 1 kb is drawn and labeled. The terminal repeat long (TR L ) region, the U L region, and an a-like sequence (double-headed arrow) are also indicated. Several MDV-1-specific ORFs neighboring the vLIP gene are displayed, as are the conserved genes U L 1 to U L 3. The gene organization of the vLIP transcript is shown in more detail below, also drawn to scale. A 500-bp bar is shown, and the TATA box, putatively used to initiate vLIP transcripts, as well as the poly(A) site and poly(A) signal, is labeled. MluI and SpeI restriction sites flanking the lipase homology region, indicated by a double-headed arrow, are also labeled. (b) The vLIP transcript as visualized by Northern blotting. In the left panel, a Northern blot was probed with a single-stranded riboprobe antisense to vLIP mRNA. RNA samples were derived from MSB-1 tumor cells which had been either treated for 24 h in the presence of sodium butyrate to induce virus replication or left untreated, as indicated. PFA was also used where indicated, to show the sensitivity of vLIP transcription to inhibition of DNA replication. To the right, RNA samples used in Northern blotting were separated on denatured agarose gels, stained with ethidium bromide, and imaged to verify that RNA integrity and quantity were comparable across all samples tested. An RNA marker (Ambion) was included to determine molecular weights.

    Article Snippet: In order to construct a shuttle vector to incorporate an in-frame deletion of amino acids 256 to 428 of the vLIP open reading frame, a 519-bp fragment was released from the pEco4.268 plasmid using MluI and SpeI, and overhangs were filled in using T4 DNA polymerase (NEB).

    Techniques: Labeling, Sequencing, Northern Blot, Derivative Assay, Inhibition, Staining, Marker

    Generation and identification of multi-transgenic PFFs and minipigs. (A) Schematic structure of tissue-specific polycistronic system (8,840 bp). The head-to-head arrows represent the primers for transgenic recognition, copy number measurement and gene expression analysis. The fragment between the two restriction sites comprises two cassettes isolated by an insulator: ( 1 ) 11β-HSD1 driven by the liver-specific PapoE; ( 2 ) hIAPP and CHOP linked to the F-2A peptide driven by the PIP. (B) PCR screening of multi-transgenic PFFs. Amplification of the PapoE-11b, PIP-CHOP, CHOP-IAPP and IAPP-pA fragments is shown, respectively. Lanes 42–44, three representative PFFs transfected by the vector. (C) Multi-transgenic piglets produced by somatic cell nuclear transfer. (D) Genomic DNA PCR identification of piglet 1 # , 2 # and negative control. F1–F3 indicate the three anticipated bands corresponding to PapoE-11b, PIP-CHOP and CHOP-IAPP, respectively. Tg, transgenic; 11β-HSD1, 11-β-hydroxysteroid dehydrogenase 1; PapoE; hIAPP; human islet amyloid polypeptide; PIP, porcine pancreas-specific insulin promoter; CHOP; C/EBP homologous protein; PCR, polymerase chain reaction; V, positive vector; N, negative control; M, 100 bp DNA ladder; W, ddH2O. MluI, Mlu I restriction enzyme site; NotI, Not I restriction enzyme site; PFFs, porcine fetal fibroblasts.

    Journal: Molecular Medicine Reports

    Article Title: Multi-transgenic minipig models exhibiting potential for hepatic insulin resistance and pancreatic apoptosis

    doi: 10.3892/mmr.2015.4582

    Figure Lengend Snippet: Generation and identification of multi-transgenic PFFs and minipigs. (A) Schematic structure of tissue-specific polycistronic system (8,840 bp). The head-to-head arrows represent the primers for transgenic recognition, copy number measurement and gene expression analysis. The fragment between the two restriction sites comprises two cassettes isolated by an insulator: ( 1 ) 11β-HSD1 driven by the liver-specific PapoE; ( 2 ) hIAPP and CHOP linked to the F-2A peptide driven by the PIP. (B) PCR screening of multi-transgenic PFFs. Amplification of the PapoE-11b, PIP-CHOP, CHOP-IAPP and IAPP-pA fragments is shown, respectively. Lanes 42–44, three representative PFFs transfected by the vector. (C) Multi-transgenic piglets produced by somatic cell nuclear transfer. (D) Genomic DNA PCR identification of piglet 1 # , 2 # and negative control. F1–F3 indicate the three anticipated bands corresponding to PapoE-11b, PIP-CHOP and CHOP-IAPP, respectively. Tg, transgenic; 11β-HSD1, 11-β-hydroxysteroid dehydrogenase 1; PapoE; hIAPP; human islet amyloid polypeptide; PIP, porcine pancreas-specific insulin promoter; CHOP; C/EBP homologous protein; PCR, polymerase chain reaction; V, positive vector; N, negative control; M, 100 bp DNA ladder; W, ddH2O. MluI, Mlu I restriction enzyme site; NotI, Not I restriction enzyme site; PFFs, porcine fetal fibroblasts.

    Article Snippet: The multiple cloning site of pcDNA3.1 (+) was digested using the Mlu I and Not I enzymes (New England Biolabs, Beijing, China), and the cytomegalovirus (CMV) promoter fragment was replaced with this synthesized fragment.

    Techniques: Transgenic Assay, Expressing, Isolation, Polymerase Chain Reaction, Amplification, Transfection, Plasmid Preparation, Produced, Negative Control

    Validation of the URMAC method by insertion (I), substitution (S), or deletion (D) of some restriction sites in pUC18 plasmid. (A) Illustration of the Modification Target (NdeI restriction site) relative to the flanking restriction sites and location of the Starter Primers SP1 and SP2. After the first PCR, the Starter DNA migrated as expected, 532 bp on a 1% agarose gel (photo, arrow at right). A 100 bp DNA size ladder is shown in left lane for comparison. (B) Diagram of the strategy for I, S, or D using the Closed Starter DNA circularized from the PCR product in (A) as template and the Opener/Mutagenic Primers . The top photo shows the PCR product, Intermediate DNA , which contained the mutations. The bottom photo shows the Modified DNA after enrichment PCR step using SP1 and SP2. (C) Validation of URMAC mutagenesis for the three different types of mutations by restriction analysis. Fig 2C shows bands of expected DNA fragment size after digestion with respective restriction enzymes. In the control Starter PCR lane, only DNA treated with NdeI enzyme, cut the DNA into two fragments of 382 150 bp. Untreated DNA or DNA treated with MluI remained at the full size of 532 bp. In the Insertion lane, both NdeI and MluI cut the DNA at the expected sizes of 382 150 for NdeI and 383 149 for MluI. In the Substitution lane, only MluI cut the DNA producing the expected 383 149 bp bands. In the Deletion lane, none of the enzymes cut the DNA, leaving the bands at their original Modified DNA size.

    Journal: PLoS ONE

    Article Title: Efficient method for site-directed mutagenesis in large plasmids without subcloning

    doi: 10.1371/journal.pone.0177788

    Figure Lengend Snippet: Validation of the URMAC method by insertion (I), substitution (S), or deletion (D) of some restriction sites in pUC18 plasmid. (A) Illustration of the Modification Target (NdeI restriction site) relative to the flanking restriction sites and location of the Starter Primers SP1 and SP2. After the first PCR, the Starter DNA migrated as expected, 532 bp on a 1% agarose gel (photo, arrow at right). A 100 bp DNA size ladder is shown in left lane for comparison. (B) Diagram of the strategy for I, S, or D using the Closed Starter DNA circularized from the PCR product in (A) as template and the Opener/Mutagenic Primers . The top photo shows the PCR product, Intermediate DNA , which contained the mutations. The bottom photo shows the Modified DNA after enrichment PCR step using SP1 and SP2. (C) Validation of URMAC mutagenesis for the three different types of mutations by restriction analysis. Fig 2C shows bands of expected DNA fragment size after digestion with respective restriction enzymes. In the control Starter PCR lane, only DNA treated with NdeI enzyme, cut the DNA into two fragments of 382 150 bp. Untreated DNA or DNA treated with MluI remained at the full size of 532 bp. In the Insertion lane, both NdeI and MluI cut the DNA at the expected sizes of 382 150 for NdeI and 383 149 for MluI. In the Substitution lane, only MluI cut the DNA producing the expected 383 149 bp bands. In the Deletion lane, none of the enzymes cut the DNA, leaving the bands at their original Modified DNA size.

    Article Snippet: Successful mutagenesis was confirmed by restriction analysis using NdeI and MluI restriction enzymes (New England Biolabs, Ipswich, MA): 2 μl of Linear Modified DNA for each mutation type was incubated with or without 5 units of restriction enzymes for 30 min at 37°C.

    Techniques: Plasmid Preparation, Modification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Mutagenesis

    Construction and characterization of MV-expressing hGCSF . ( a ) Schematic showing cloning of DNA encoding human cytokine granulocyte colony-stimulating factor (hGCSF) p (+) MV-NSe upstream of M, using AatII and MluI restriction enzymes. ( b ) One step growth

    Journal: Molecular Therapy

    Article Title: The Role of Neutrophils in Measles Virus–mediated Oncolysis Differs Between B-cell Malignancies and Is Not Always Enhanced by GCSF

    doi: 10.1038/mt.2015.149

    Figure Lengend Snippet: Construction and characterization of MV-expressing hGCSF . ( a ) Schematic showing cloning of DNA encoding human cytokine granulocyte colony-stimulating factor (hGCSF) p (+) MV-NSe upstream of M, using AatII and MluI restriction enzymes. ( b ) One step growth

    Article Snippet: It was then PCR amplified with the following primers, forward primer MluI+hGCSF: 5′- agtattac acgcgt atggctggacctgccacccagagc-3′ and reverse primer: AatII_hGCSF: 5′-tacagtcg gacgtc attcagggctgggcaaggtggcg-3′, and the PCR product was cloned into the MVNSe backbone using AatII and MluI restriction endonucleases (New England Biolab, UK), replacing GFP, upstream of MV M gene.

    Techniques: Expressing, Clone Assay

    Electrophoresis of six constructs digested with Mlu I and Xho I. M1: 100 bp DNA ladder marker, M2: 1 kb DNA ladder marker, Lane1: pCOLH 1 0.1, lane2: pCOLH 1 0.27, lane3: pCOLH 1 0.5, Lane4: pCOLH 1 0.9, Lane5: pCOLH 1 1.5, Lane6: pCOLH 1 2.5. Six recombinant plasmids

    Journal:

    Article Title: Transcriptional regulation of human α1(I) procollagen gene in dermal fibroblasts

    doi: 10.3748/wjg.v10.i10.1447

    Figure Lengend Snippet: Electrophoresis of six constructs digested with Mlu I and Xho I. M1: 100 bp DNA ladder marker, M2: 1 kb DNA ladder marker, Lane1: pCOLH 1 0.1, lane2: pCOLH 1 0.27, lane3: pCOLH 1 0.5, Lane4: pCOLH 1 0.9, Lane5: pCOLH 1 1.5, Lane6: pCOLH 1 2.5. Six recombinant plasmids

    Article Snippet: The PCR products were digested with Mlu I and Xho I and then ligated to M luI- Xho I linearized pCAT3 vector with T4 DNA ligase (Promega).

    Techniques: Electrophoresis, Construct, Marker, Recombinant

    Specificity of telomere interactions analyzed by the 3C procedure. ( A ) Physical map of pLUS892L and the predicted cross-linked, ligated products. On pLUS892L (top), the positions of the L and R primers and their distances to the nearest MluI (‘Ml’) sites are indicated (in kb). On the expected intramolecular (middle) and intermolecular (bottom) products, the expected PCR products and their sizes (in kb) are indicated. Cross-linking is depicted by the dashed arcs. The other symbols are as in Figure 1 . ( B ) Detection of intermolecular and intramolecular cross-linking. S. lividans 3200/pLUS892L mycelium was treated with DSS, and lysed by protoplasting and osmotic shock. DNA in the lysate was digested with MluI, and ligated at two different concentrations (top panel, 20-µl ligation reaction; bottom panel, 1-ml ligation reaction) followed by LM-PCR using the R, L or both primers (‘R + L’) in the absence (‘0%’) or presence (‘10%’) of DMSO. The PCR products were analyzed by gel electrophoresis. The sizes of the PCR products are indicated (in kb). ( C ) Detection of intramolecular cross-linking after GnHCl centrifugation. Cross-linking and detection were performed as in B except for an additional GnHCl–CsCl gradient centrifugation step (right half) DNA before cross-linking. The volume of the ligation reaction was 1 ml.

    Journal: Nucleic Acids Research

    Article Title: Linear Streptomyces plasmids form superhelical circles through interactions between their terminal proteins

    doi: 10.1093/nar/gkq1204

    Figure Lengend Snippet: Specificity of telomere interactions analyzed by the 3C procedure. ( A ) Physical map of pLUS892L and the predicted cross-linked, ligated products. On pLUS892L (top), the positions of the L and R primers and their distances to the nearest MluI (‘Ml’) sites are indicated (in kb). On the expected intramolecular (middle) and intermolecular (bottom) products, the expected PCR products and their sizes (in kb) are indicated. Cross-linking is depicted by the dashed arcs. The other symbols are as in Figure 1 . ( B ) Detection of intermolecular and intramolecular cross-linking. S. lividans 3200/pLUS892L mycelium was treated with DSS, and lysed by protoplasting and osmotic shock. DNA in the lysate was digested with MluI, and ligated at two different concentrations (top panel, 20-µl ligation reaction; bottom panel, 1-ml ligation reaction) followed by LM-PCR using the R, L or both primers (‘R + L’) in the absence (‘0%’) or presence (‘10%’) of DMSO. The PCR products were analyzed by gel electrophoresis. The sizes of the PCR products are indicated (in kb). ( C ) Detection of intramolecular cross-linking after GnHCl centrifugation. Cross-linking and detection were performed as in B except for an additional GnHCl–CsCl gradient centrifugation step (right half) DNA before cross-linking. The volume of the ligation reaction was 1 ml.

    Article Snippet: Five microliter of the lysate was digested with MluI and ligated with T4 DNA ligase (TaKaRa Bio Inc.) in a final volume of 20 µl.

    Techniques: Polymerase Chain Reaction, Ligation, Nucleic Acid Electrophoresis, Centrifugation, Gradient Centrifugation

    In vitro cross-linking of telomeres. ( A ) Without GnHCl treatment. Total DNA of S. lividans 3200/pLUS891L was treated with DSS or DSG, digested with BclI (Bc) or BglII (Bg), fractionated by gel electrophoresis and hybridized with pLUS891L DNA probe. ( B ) With GnHCl treatment. Genomic DNA of S. lividans 3200/pLUS892L was isolated by GnHCl–CsCl gradient centrifugation and treated with DSS or DSG. The treated DNA was digested with BclI or MluI, fractionated by gel electrophoresis and hybridized to the SCP1 terminal DNA probe. The physical map of pLUS892L is shown above with the BclI and MluI (Ml) sites and fragments sizes indicated. In the control experiment (right panel), the samples were treated with proteinase K before electrophoresis. The cross-linked DNA is indicated by an asterisk.

    Journal: Nucleic Acids Research

    Article Title: Linear Streptomyces plasmids form superhelical circles through interactions between their terminal proteins

    doi: 10.1093/nar/gkq1204

    Figure Lengend Snippet: In vitro cross-linking of telomeres. ( A ) Without GnHCl treatment. Total DNA of S. lividans 3200/pLUS891L was treated with DSS or DSG, digested with BclI (Bc) or BglII (Bg), fractionated by gel electrophoresis and hybridized with pLUS891L DNA probe. ( B ) With GnHCl treatment. Genomic DNA of S. lividans 3200/pLUS892L was isolated by GnHCl–CsCl gradient centrifugation and treated with DSS or DSG. The treated DNA was digested with BclI or MluI, fractionated by gel electrophoresis and hybridized to the SCP1 terminal DNA probe. The physical map of pLUS892L is shown above with the BclI and MluI (Ml) sites and fragments sizes indicated. In the control experiment (right panel), the samples were treated with proteinase K before electrophoresis. The cross-linked DNA is indicated by an asterisk.

    Article Snippet: Five microliter of the lysate was digested with MluI and ligated with T4 DNA ligase (TaKaRa Bio Inc.) in a final volume of 20 µl.

    Techniques: In Vitro, Nucleic Acid Electrophoresis, Isolation, Gradient Centrifugation, Electrophoresis

    CRISPR-Cas9-mediated crh gene disruption in CRH PVN neurons of CRH::Cre-Cas9 mice. (A) Generation of CRH::Cre-Cas9 mice. (B) Schematic of AAV sgCRH12 vector design. (C) Schematic of AAV-DJ-U6- sgControl/sgCRH12-EF1α-DIO-mCherry to unilateral PVN of CRH::Cre-Cas9 mice (n = 3, AP: −0.90 mm, ML: ±0.30 mm, DV: −4.5 + -4.8 mm, 0.3 μl each depth). (D) Representative slices from CRH::Cre-Cas9 mice injected with AAV vectors carrying sgControl or sgCRH12. Note the intact CRH staining on the contralateral side without viral infection in the sgCRH12 panel.

    Journal: bioRxiv

    Article Title: Hypothalamic circuitry underlying stress-induced insomnia and peripheral immunosuppression

    doi: 10.1101/2020.04.29.069393

    Figure Lengend Snippet: CRISPR-Cas9-mediated crh gene disruption in CRH PVN neurons of CRH::Cre-Cas9 mice. (A) Generation of CRH::Cre-Cas9 mice. (B) Schematic of AAV sgCRH12 vector design. (C) Schematic of AAV-DJ-U6- sgControl/sgCRH12-EF1α-DIO-mCherry to unilateral PVN of CRH::Cre-Cas9 mice (n = 3, AP: −0.90 mm, ML: ±0.30 mm, DV: −4.5 + -4.8 mm, 0.3 μl each depth). (D) Representative slices from CRH::Cre-Cas9 mice injected with AAV vectors carrying sgControl or sgCRH12. Note the intact CRH staining on the contralateral side without viral infection in the sgCRH12 panel.

    Article Snippet: Amplified fragments were cloned tandemly into MluI-digested pAAV EF1α DIO mCherry (Addgene plasmid 20299) by Gibson assembly method.

    Techniques: CRISPR, Mouse Assay, Plasmid Preparation, Injection, Staining, Infection